The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate

Abstract

Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.

FIG. 1

SDS-PAGE analysis of purified radiolabeled virus particles; 5.0 × 10⁴ cpm of virus was loaded in each lane. Molecular mass markers (kDa) are shown in the extreme right-hand and left-hand lanes.

FIG. 2

Binding of radiolabeled purified viruses to CHO cells. The bars represent averages of triplicate binding assays with 10⁵ cpm of virus and ∼10⁶ cells per reaction. Each set of triplicates was repeated at least twice. The error bars represent standard deviations.

FIG. 3

Binding of viruses to CHO cells without (solid bars) or with (hatched bars) previous digestion with heparinase I. The cells were either digested with 8 U of heparinase I (in phosphate-buffered saline with 0.1% BSA/ml) or mock-digested (phosphate-buffered saline with 0.1% BSA only) for 1 h at 37°C followed by processing for attachment assays as described in Materials and Methods. The bars represent averages of triplicate binding assays with 10⁵ cpm of virus and ∼10⁶ cells per reaction. Each set of triplicates was repeated at least twice. The error bars represent standard deviations.

FIG. 4

Binding of radiolabeled viruses prepared in BHK cells or C6/36 cells to CHO cells. The bars represent averages of triplicate binding assays with 10⁵ cpm of virus and ∼10⁶ cells per reaction. Each set of triplicates was repeated at least twice. The error bars represent standard deviations.